JP2000287689A - Screening of substance which suppresses or promotes apoptosis - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for screening a substance which suppresses or promotes apoptosis by incubating VDAC-liposome, a VDAC-passing indicator, and a subject substance, followed by detecting a change in the concentration of the indicator. SOLUTION: A method is provided for screening a substance which suppresses or promotes apoptosis by determining the presence or absence of the action of suppressing or promoting the apoptosis of a subject substance by incubating VDAC-liposome obtained by reconstituting VDAC in a small unilamellar vesicle, phosphor-labelled cytochrome c, and/or an indicator which can pass VDAC such as isotope-labelled sucrose, and a subject substance such as protein and polypeptide, followed by detecting a change in the concentration of the indicator inside and outside VDAC-liposome before and after incubation.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for screening a substance having an apoptosis (cell death) inhibitory action and an apoptosis promoting action, more specifically, a method for screening an apoptosis inhibitory substance or an apoptosis promoting substance using VDAC-liposome, The present invention relates to a novel polypeptide having an apoptosis-suppressing action and an apoptosis-promoting action.

[0002]

2. Description of the Related Art Apoptosis is cell death caused by cells under physiological conditions. The form of apoptosis involves chromosome aggregation of cell nuclei, fragmentation of cell nuclei, loss of cell surface microvilli, and condensation of cytoplasm. Characteristically, recently, a cell surface receptor Fas antigen that induces apoptosis, a protease (caspase) essential for inducing apoptosis, an oncogene bcl-2 gene that suppresses apoptosis, and the like have been elucidated.

[0003] The oncogene bcl-2 gene has a chromosomal translocation t (14; 18) (q21; q) associated with follicular lymphoma.
32) identified as an oncogene activated by
A unique oncogene with apoptosis suppressing function, a number of proteins that exhibit homology to the Bcl-2 is referred to as Bcl-2 family, the Bcl-2 family, in Bcl-2 and Bcl-x _L It is known that there are representative members having an apoptosis-suppressing function and members having an apoptosis-promoting (or inducing) function represented by Bax and Bak.

In addition, mitochondria play a very important role in transducing apoptotic signals,
The apoptotic changes in the mitochondria adjusted Bcl-2 family proteins, the Bcl-2 and Bcl-x _L having a apoptosis-suppressing function to inhibit mitochondrial membrane potential ([Delta] [Psi]) loss and cytochrome c release (Proc. Natl. Acad . Sci. USA 95, 14681-14686,
1998. J. Exp. Med. 183, 1533-1544, 1996.Cell 91,
627-637, 1997. Proc. Natl. Acad. Sci. USA 95, 1455-
1459) and Bax and B having apoptosis promoting function
ak induces mitochondrial membrane potential (ΔΨ) loss and cytochrome c release (Proc. Natl. Acad. Sci.
USA 95, 4997-5002, 1998. J. Cell Biol. 143, 217-22
4, 1998. Proc. Natl. Acad. Sci. USA 95, 14681-1468
6, 1998) is also known.

[0005] A polyprotein channel called a membrane permeability transition (hereinafter referred to as "PT") pore mediates a Bax- and Bak-dependent change in membrane permeability (Pr).
oc.Natl. Acad. Sci. USA 95, 4997-5002, 1998. Proc.
Natl. Acad. Sci. USA 95, 14681-14686, 1998. Scienc
e 281, 2027-2031, 1998) and polyprotein channels are thought to consist of VDAC (voltage-dependent anion channel), also called mitochondrial porin, adenine nucleotide translocator (ANT), cyclophilin D and several other molecules. (J. B.
ioenerg. Biomembr. 26, 509-517, 1994. Biochem. Bio
phys. Acta 1241, 139-176, 1995) are also known.

[0006] VADCs are small proteins abundant in the outer membrane of mitochondria and form large (2.6 nm) voltage-dependent pores when incorporated into planar lipid bilayers to form VADC-liposomes. Is known (J. Membr. Biol. 111, 103-111, 198
9) In addition, VDAC is considered to function physiologically as a pathway for various substances including ions and intermediate metabolites to enter and exit mitochondria. Furthermore, the present inventors have recently shown that Bax and Bak interact with PT pores (Pro
c. Natl. Acad. Sci. USA 95, 14681-14686, 1998. Sci.
ence 281, 2027-2031, 1998). Bax is ANT
(Adenine nucleotide translocator) has also been reported to sensitize ANT and PT pore liposomes to atractylic acid, an ANT ligand (Science 281, 2027-2031, 1998).

[0007]

Bcl-2 and Bax are deeply involved in carcinogenesis. follicular lymphoma and chronic large cell lymphoma with t (14; 18) translocation, t (1
8; 22) (q21; q11) or t (2; 18)
In chronic lymphocytic leukemia having (q11; q21), Bcl-2 is deregulated in its expression by translocation to the immunoglobulin heavy chain and light chain regions, and promotes cell death suppression and contributes to carcinogenesis. Bax, on the other hand, functions as a tumor suppressor gene and is frequently used in blood cell lineage tumor cell lines.
Mutations in the genes (BH1 and BH3 regions) have been observed, suggesting involvement in human tumors.

[0008] Bcl-2 is deeply involved in neurodegenerative diseases. SMN (survival motor neuron) that binds to Bcl-2 and enhances its apoptosis-suppressing function
Protein is a causative gene product of spinal muscular atrophy, a genetic disease accompanied by motor neuron degeneration, and a decrease in Bcl-2 function due to SMN deficiency is closely related to the development of this disease .

An object of the present invention is to provide an apoptosis-suppressing substance or an apoptosis-promoting substance that can be expected to be used as a drug or a diagnostic agent, since the Bcl-2 family having an apoptosis-suppressing action or an apoptosis-promoting action is closely related to various diseases. And to provide an apoptosis-suppressing substance or an apoptosis-promoting substance.

[0010]

The present inventors have SUMMARY OF THE INVENTION may, functions and suppressing apoptosis by Bcl-2, Bcl-x _L belonging to the Bcl-2 family proteins that regulate apoptosis, Bax, apoptosis due Bak Although it has been shown that the promoting function is performed through mitochondria, intensive studies on its target molecule and the use of a liposome system
The present inventors have found that the 1-2 family protein regulates cytochrome c release and ΔΨ loss, which are central phenomena in apoptosis signaling, by directly targeting the protein VDAC of the outer layer of the mitochondrial membrane. Reached.

That is, the present invention provides a method for incubating a VDAC-liposome, an indicator substance capable of passing through the VDAC, and a test substance, and detecting a change in the concentration of the indicator substance inside and outside the VDAC-liposome before and after the incubation. A method for screening for an apoptosis-suppressing substance or an apoptosis-promoting substance, which comprises determining the presence or absence of an apoptosis-suppressing action or an apoptosis-promoting action of a test substance (claim 1); The method for screening for an apoptosis-suppressing substance or apoptosis-promoting substance according to claim 1 or claim 2, wherein the incubation is carried out in a state where the test substance is present in the VDAC-liposome. Specially The screening method for an apoptosis-suppressing substance or an apoptosis-promoting substance according to claim 1 or 2 (claim 3), and the incubation is performed in a state where the apoptosis-suppressing substance or the apoptosis-promoting substance is present together with the test substance. A screening method for an apoptosis-suppressing substance or an apoptosis-promoting substance according to claim 1 or claim 2 (claim 4);
The method for screening an apoptosis-suppressing substance or apoptosis-promoting substance according to any one of claims 1 to 4, wherein the AC-liposome is a VDAC-liposome prepared using recombinant human VDAC (claim 5). Or the VDAC-liposome is a VDAC-liposome obtained by reconstituting VDAC into small unilamellar vesicles, and the method for screening an apoptosis-suppressing substance or apoptosis-promoting substance according to any one of claims 1 to 5 (claim). 6) The method for screening an apoptosis-suppressing substance or apoptosis-promoting substance according to any one of claims 1 to 6, wherein the indicator substance is a labeling compound, and the labeling compound is a fluorescent label. It is cytochrome c and / or isotope-labeled sucrose. Screening method for apoptosis-suppressing or -promoting substance described for (claim 8).

The present invention also provides a method for screening an apoptosis-suppressing substance or an apoptosis-promoting substance according to any one of claims 1 to 8, wherein the test substance is a protein or a polypeptide (claim 9). 10. The protein or polypeptide according to claim 9, wherein the protein or polypeptide has an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence of the Bcl-2 family protein. A screening method for an apoptosis-suppressing substance or an apoptosis-promoting substance (Claim 10), and a method in which a protein or polypeptide has one or several Amino acids are deleted, substituted or added The method for screening for an apoptosis-suppressing substance or apoptosis-promoting substance according to claim 9, wherein the protein or polypeptide is a protein or polypeptide containing the amino acid sequence described above. A protein or polypeptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in a polypeptide having an amino acid sequence of the fourth to 23rd amino acids in the amino acid sequence to be obtained. 9. The method for screening an apoptosis-suppressing substance or an apoptosis-promoting substance according to claim 9 (claim 12);
The apoptosis-inhibiting substance according to claim 9, which is a protein or polypeptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the polypeptide having the 74th to 74th amino acid sequence. Or a method for screening an apoptosis-promoting substance (Claim 13), wherein the protein or polypeptide is a DN encoding a Bcl-2 family protein.
10. The apoptosis-inhibiting substance or apoptosis according to claim 9, which is a protein or polypeptide obtained by using as a probe a DNA comprising the nucleotide sequence of A or its complementary sequence and a part or all of these sequences. A screening method for a promoting substance (claim 1
4) Alternatively, as a probe, a protein or polypeptide is a probe comprising a base sequence from the 32nd to the 751st base or a complementary sequence thereof and a DNA containing a part or all of these sequences in the base sequence shown in SEQ ID NO: 1. 10. The method for screening an apoptosis-suppressing substance or apoptosis-promoting substance according to claim 9, comprising a protein or a polypeptide obtained by using the method.
5) Alternatively, when the protein or polypeptide is a probe comprising a nucleotide sequence represented by SEQ ID NO: 3 from the 135th to 836th nucleotides or a complementary sequence thereof and a DNA containing a part or all of these sequences. 10. The method for screening for an apoptosis-suppressing substance or an apoptosis-promoting substance according to claim 9, wherein the protein or polypeptide is obtained by using the protein or polypeptide.
Out of the base sequence shown in 163 to 222 or a complementary sequence thereof and a protein or polypeptide obtained by using a DNA containing a part or all of these sequences as a probe. A method for screening an apoptosis-suppressing substance or apoptosis-promoting substance according to claim 9 (claim 17).

Further, the present invention relates to a polypeptide having the 7th to 30th amino acid sequence of the amino acid sequence shown in SEQ ID NO: 2 (claim 18), and a polypeptide having the amino acid sequence shown in SEQ ID NO: 2 One or several amino acids are deleted in the 30th to 30th amino acid sequences,
A polypeptide having at least a substituted or added amino acid sequence and having an apoptosis-suppressing activity (Claim 19), and a polypeptide having an amino acid sequence of the 4th to 23rd amino acids of the amino acid sequence shown in SEQ ID NO: 4 (Claim 19) Item 20) and an apoptosis-suppressing effect, which comprises at least an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence from the 4th to 23rd amino acids in the amino acid sequence shown in SEQ ID NO: 4. Of the polypeptide (claim 21) or the amino acid sequence shown in SEQ ID NO: 6 from position 55 to position 7
A polypeptide having a fourth amino acid sequence (claim 2
2) or 5 of the amino acid sequence shown in SEQ ID NO: 6
A polypeptide having at least one amino acid sequence in which one or several amino acids have been deleted, substituted or added in the fifth to 74th amino acid sequence and which has an apoptosis-promoting action (claim 23); The polypeptide according to any one of claims 18 to 23 (claim 24), which is chemically modified for introduction, and apoptosis obtained by the screening method according to any one of claims 1 to 17. Inhibitory substance (Claim 2
5) or an apoptosis-promoting substance obtained by the screening method according to any one of claims 1 to 17 (claim 26)
About.

[0014]

BEST MODE FOR CARRYING OUT THE INVENTION The VDAC-liposome in the present invention is a VDAC-liposome,
Any type of DAC may be used as long as it has a structure embedded in the lipid bilayer of a liposome, which is a closed vesicle composed of a lipid bilayer. The VDAC constituting the VDAC-liposome is not limited in its origin. VDAC-liposome prepared using a human-derived VDAC such as a recombinant human VDAC is preferable from the viewpoint of developing a drug or the like, and VDAC-As liposomes, small unilamellar vesicles (SUVs)
Is preferred from the viewpoint of reaction sensitivity. As a method for preparing the liposome, a conventionally known method can be used.

As the indicator substance in the present invention, any substance can be used as long as it can pass through the VDAC channel in the presence of an apoptosis-promoting substance or the like. In determining the presence or absence of, it is preferable to easily detect a change in concentration before and after incubation, and include a labeling compound, a dye, an enzyme, and the like. Cytochrome c whose release is considered to be a central phenomenon in transduction of apoptotic signals
Can be specifically exemplified by labeled cytochrome c in which is modified by a labeling compound such as a fluorescent substance. When the indicator substance is incubated with the VDAC-liposome, one or more of them may be used simultaneously,
In addition, it can be present inside the VDAC-liposome and / or outside the VDAC-liposome.

The test substance in the present invention may be any substance as long as it is expected to determine the presence or absence of an apoptosis-suppressing action or an apoptosis-promoting action. High protein can be specifically exemplified. Among them, a modified or modified protein or modified or modified polypeptide of a protein or polypeptide consisting of all or a part of a Bcl-2 family protein already known as a regulator of apoptosis Is preferred. As modified or altered proteins and polypeptides, for example,
Protein or polypeptide having one or more amino acids deleted, substituted or added in the amino acid sequence of the protein or polypeptide, the nucleotide sequence of DNA encoding the protein or polypeptide, or a part of these sequences Or a protein or polypeptide obtained by using a DNA containing the whole as a probe.

As a Bcl-2 family protein, a Bcl-2 protein having the amino acid sequence shown in SEQ ID NO.
1-2, a B having the amino acid sequence of SEQ ID NO: 4
_{cl-x L, Bcl-w} , Mcl-1, A1 (Bfl-
1), BHRF-1 (Epstein-Barr vi)
rus), LMW-5-HL (African swi)
ne five virus), E1B19kDa
(Adenovirus), Ksbcl-2 (HHV
8), ORF16 (herpesvirus saim)
The iri), as a protein having an apoptotic effect, Bax having the amino acid sequence shown in SEQ ID NO: _{6, Bcl-x S, Bad} , Bak, Mtd (Bo
k), Diva, Bik, Bid, Bim, Hrk (D
P5), Blk, Bnip3, and Bnip3L. In addition, the polypeptide comprising a part of the Bcl-2 family protein is a polypeptide having an apoptosis-suppressing action,
Bc consisting of the 7th to 30th amino acid sequence described above
The Bcl-x _L derived polypeptide consisting fourth from 23 amino acid sequence of the polypeptide and SEQ ID NO: 4, wherein from l-2, as a polypeptide having an apoptotic effect, a 55 th of SEQ ID NO: 6, wherein Bax-derived polypeptide consisting of the 74th amino acid sequence,
Each can be specifically exemplified.

Further, in the method for screening an apoptosis-suppressing substance or apoptosis-promoting substance of the present invention, the protein or polypeptide may be a base sequence of a DNA encoding a Bcl-2 family protein or a sequence complementary thereto and a sequence of these sequences. D including part or all
A protein or polypeptide obtained by using NA as a probe can also be advantageously used as a test substance. Then, the DNA encoding the Bcl-2 family proteins, for example, DNA encoding the Bcl-2 shown in SEQ ID NO: 1, DNA encoding the Bcl-x _L as shown in SEQ ID NO: 3, SEQ ID NO: 5 The DNA encoding Bax can be specifically exemplified. As a part of the nucleotide sequence of the DNA encoding the Bcl-2 family protein, for example, the nucleotide sequence from the 32nd to 751 Up to the base sequence, one of the base sequences shown in SEQ ID NO: 3
Specific examples include the base sequence from the 35th position to the 836th position, and the base sequence from the 163rd position to the 222nd position in the base sequence shown in SEQ ID NO: 5.

The test substance can be used alone or in combination with one or more known apoptosis-suppressing substances or one or more apoptosis-promoting substances when incubating with the VDAC-liposome. When the test substance and the apoptosis-suppressing substance are used together, the apoptosis-promoting substance can be screened. When the test substance and the apoptosis-promoting substance are used together, the apoptosis-suppressing substance can be screened. Substances that antagonize the promoter can also be screened. When used in combination, the test substance and the apoptosis-suppressing substance or the apoptosis-promoting substance are preferably present in a VDAC-liposome.
It can also be present outside the liposome. And
Conventionally known methods for allowing a test substance and an apoptosis-suppressing substance or an apoptosis-promoting substance, a test substance and an indicator substance, a test substance, an indicator substance, an apoptosis-suppressing substance, or an apoptosis-promoting substance to be present in a VDAC-liposome during the incubation. Can be used.

In the method for screening an apoptosis-suppressing substance or apoptosis-promoting substance of the present invention, conditions such as temperature, pH, reaction time and the like in the incubation are not particularly limited, but the indicator substance inside and outside the VDAC-liposome before and after the incubation. It is preferable to select a condition under which the change in concentration can be detected with high sensitivity.

The present invention also relates to an amino acid sequence represented by SEQ ID NO: 2 from the 7th to the 30th amino acid sequence;
2 to 4 from the amino acid sequence shown in SEQ ID NO: 4
A polypeptide having the 55th to 74th amino acid sequence of the third amino acid sequence or the amino acid sequence of SEQ ID NO: 6, or the 7th to 30th amino acid sequence or sequence of the amino acid sequence of SEQ ID NO: 2 In the amino acid sequence of the 4th to 23rd amino acids in the amino acid sequence of No. 4 or the 55th to 74th amino acid sequence of the amino acid sequence of SEQ ID NO: 6, 1
Alternatively, polypeptides having an apoptosis-controlling action including at least an amino acid sequence in which several amino acids have been deleted, substituted or added, polypeptides in which these peptides have been chemically modified for introduction into cells, The present invention relates to an apoptosis-suppressing substance or an apoptosis-promoting substance obtained by a screening method for an apoptosis-suppressing substance or an apoptosis-promoting substance. Among them, the 7th to 30th amino acid sequence of the amino acid sequence shown in SEQ ID NO: 2, the 4th to 23rd amino acid sequence of the amino acid sequence shown in SEQ ID NO: 4 or the amino acid shown in SEQ ID NO: 6 From the 55th to 74th of the sequence
The third amino acid sequence is known, but a polypeptide consisting of these amino acid sequences has never been newly prepared. Even more, a polypeptide consisting of these amino acid sequences acts on a VDAC channel, thereby inhibiting apoptosis or promoting apoptosis. It is the beginning of the present invention that the present invention can be exhibited. Examples of the polypeptide that has been chemically modified for introduction into cells include a polypeptide to which an oligopeptide derived from the protein TAT produced by the HIV virus, which facilitates the transfer into cells, is included.

[0022]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described in detail below with reference to embodiments, but the technical scope of the present invention is not limited to these embodiments. [Materials and Methods] (Acquisition of Antibody) An anti-cytochrome c monoclonal antibody cross-reacting with rat and horse cytochrome c (7H8.
2C12), anti-yeast cytochrome c polyclonal antibody and anti-rat ANT antibody were obtained from Dr. E. Margoliash, respectively.
(University of Illinois, Illinois), Dr. G. Shatz (University of Basel, Switzerland) and Dr. HH Schmid (University of Minnesota,
Minnesota) was used. Rat VDA
The anti-human VDAC (porin) monoclonal antibody (31HL) that cross-reacts with C is available from Calbioch (Calbioch).
em, La Jolla, CA), anti-human Bcl-x _L polyclonal antibody (L19) and anti-human Bax polyclonal antibody (N20) were obtained from Santa Cruz Biotechology, Santa Cruz, CA, respectively. .

(Immunoprecipitation reaction and Western blot analysis) Rat liver mitochondria were isolated from the literature (Proc. Natl.
Acad. Sci. USA 95, 1455-1459, 1998). Lysis buffer containing proteinase inhibitor (Hepes, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid at final concentration of 10 mM)
pH 7.4, 5 mM KCl, 5 mM MgCl ₂ , 1
Using citrated cells and mitochondria dissolved in mM EGTA (ethylene glycol bis (2-aminoethyl ether) tetraacetic acid, 0.5% NP40) and mitochondria, the literature (Proc. Natl. Acad. Sci. USA 95, 14681-1
4686) as described. To perform immunoprecipitation experiments using liposomes, the literature (Mol. Cell 1,
337-346, 1998), DTBP was used as a protein crosslinker. To detect binding between purified proteins, GST-
Bcl-x _L (0.2 μg) or GST (0.2 μg)
Using either VDAC (0.2 μg) and AN
T (0.2 μg) was incubated for 3 hours. Then, using a glutathione (GSH) -Sepharose gel or a Sepharose gel, these proteins were incubated and adsorbed for 3 hours. After brief centrifugation, the gel was washed three times with lysis buffer and resuspended in sample buffer for SDS (sodium dodecyl sulfate) -PAGE.

(Peptide sequencing) The immunoprecipitate was subjected to SDS.
After electrophoresis on a polyacrylamide gel, the separated proteins were cut out and digested with lysyl endopeptidase at 35 ° C. for 1 day. The obtained peptide fragment was separated using a TSK gel (“ODS-80Ts” manufactured by Tosoh), and microsequencing was performed directly from the N-terminal side by Edman degradation to determine the peptide sequence.

[0025] (purification of the protein) Human Bcl-x _L,
Two Bcl-x _L mutant and human Bak (C-terminus 2
Are we reforming missing) a one amino acid residue, E. coli (Escheric
hia coil ) was expressed as a GST fusion protein in the DH5α strain and purified using a glutathione-Sepharose column. Thrombin was allowed to act on the purified GST fusion protein to separate it from GST. Human Bax is H
Literature (Proc. Natl. Acad. Sc
i. USA 95, 14681-14686). Using Express System (Invitrogen)
HA-tagged human VDAC1 was expressed in Escherichia coli strain DE3, and purified with an HA (dry hydroxyapatite) column. GST or His from empty vector
A mock control protein was prepared using the tag protein.

References (Biochem. Biophys. Acta 905, 499-
502, 1987), rat liver VDAC was purified. That is, rat liver mitochondria were reduced to 10 mM.
Tris-HCl buffer (pH 7.0), 1 mM EDT
A and 3% Triton-X100 were suspended at 0 ° C. for 30 minutes and separated by ultracentrifugation at 37,000 g for 45 minutes. The supernatant was added to an HA and Celite (9: 1) column (3 g) and the first eluate (1 ml) was collected from the fractions as purified VDAC protein. The purified VDAC showed one band on the gel by SDS polyacrylamide electrophoresis. r
The VDAC purity was 95% or higher. Rat heart AN
T was purified as described in the literature (FEBS lett. 426, 97-101, 1998). The purity of the purified ANT was determined by a method similar to the above, and was found to be 90% or more.

(Reconstitution of VDAC in liposome) The ultrasonic freeze-thaw method described in the literature (Biochem. Biopys. Acta 1145, 168-176, 1993) was slightly modified and purified in small unilamellar vesicles. VDAC and recombinant VDA
C was reconstituted (Biochem. Biopys. Acta 1145, 168-17).
6, 1993). That is, in 1 ml of a liposome medium containing 30 mM sodium sulfonate and 20 mM tricine-NaOH (pH 7.3 or 5.2), in the presence or absence of 80 mM sucrose, 100 mg of phospholipid (soybean, type II-S) ) Was dissolved. After sonication, purified VDAC protein or recombinant VDAC
The protein (200 μg) was added to rBcl-x _L (200
μg) and rBax (200 μg) in the presence or absence of 1 ml of phospholipid and the suspension was subjected to two freeze-thaw cycles. The heat-denatured VDAC protein was obtained by heating at 95 ° C. for 10 minutes. Produce unilamellar VDAC liposomes by mild sonication,
The product was fractionated on a Sephadex G-50 column. The light scattering of each fraction was measured at a wavelength of 520 nm with a spectrophotometer (manufactured by Shimadzu Corporation; UV-160A), and the light scattering was measured at an arbitrary 70 nm.
Fractions containing 0-900 units / μl were used for the experiments. Plain liposomes were produced in a similar manner without any added protein.

A sucrose uptake experiment was performed while measuring ¹⁴ C sucrose absorption. The liposomes (20 μl) were incubated with 5 μl of ¹⁴ C sucrose (97%; 200 μCi / ml) at 25 ° C.
Free ¹⁴ C sucrose was removed by centrifugation using a 000 restriction filter (Millipore; Ultrafree-MC 30,000). ¹⁴ C sucrose incorporated in the collected liposomes was measured using a gamma scintillation counter (“1414” manufactured by Wallac).

(Cytochrome c transition experiment) Reference (Wiley Inte
rscience. 5.3.1-5.3.11, 1990), fluorescein isothiocyanate (hereinafter referred to as “FITC”) labeled cytochrome c was prepared. That is, 0.05
1 mM horse cytochrome c was dialyzed against a labeling buffer (pH 9.2) containing M borate and 0.2 M sodium chloride. 2 with FITC (250 μg)
After a period of incubation, unbound FITC was removed by dialysis against a labeling buffer followed by Sephadex G-25 column chromatography. The molar ratio of FITC / cytochrome c measured with a spectrophotometer was 0.47. FITC-labeled cytochrome c was detected using a lumino image analyzer ("LAS-1000" manufactured by Fuji Film Co., Ltd.). Liposomes (2
μl) together with both sucrose (50 mM) and FITC-labeled cytochrome c (50 μM), rBax (1.5
μg) together with rBax and rBcl-x _L (both 1.5
μg) with or without both.
9 μl, incubated at pH 5.2, and labeled cytochrome c was visualized using a confocal microscope (LSM-745; Olympus).

Next, a cytochrome c leakage experiment was performed by the following method. Cytochrome c (100 μM) was mixed with liposome (1 ml) buffer containing sucrose (80 mM) and subjected to two freeze-thaw cycles. After unilamellarization, 1 ml sucrose-free buffer with or without 20 μg / ml rBak for 10 minutes at 25 ° C.
0 μl of the liposomes were incubated. Next, to separate liposomes from free cytochrome c, M.
W. Separated by centrifugation using a 30,000 restriction filter. The amount of free cytochrome c was determined using a spectrophotometer at an absorbance of 408 nm and the total cytochrome c was determined.
Was calculated.

(Yeast Mitochondria) A VDAC-deficient yeast strain (hereinafter referred to as "ΔVDAC"; M22-2) and its parent strain (M3) are Dr. Forte (Dr. M. Forte, Volum Institute for Advanced Biochemical Research, Oregon Vollum Institute for A
Those provided by Advanced Biomedical Research, Oregon) were used. By transfecting the human vdac1 cDNA with lithium acetate,
A ΔVDAC yeast strain expressing DAC1 was created. 2 mg / g cells to form spheroplasts
Using zymolyase 20T from J. Biol. Chem.
257, 13028-13033, 1982), and mitochondria were isolated. Cephaloplasts were homogenized using a tight fitting homogenizer. 0.6 M mannitol, 10 mM TrisCl
The isolated mitochondria were suspended in (pH 7.4) and 0.1% fatty acid-free BSA (hereinafter referred to as “yMt buffer”). Mitochondria (0.5 mg / m2) in yMT buffer supplemented with 4.2 mM succinate
l) was incubated at 25 ° C. Literature (Proc. Nat
Δcad was measured by measuring the absorption of rhodamine 123 (Rh123) as described in l. Acad. Sci. USA 95, 1455-1459). The above-mentioned mitochondria were spun, and the resulting supernatant was detected for cytochrome c release by Western blot analysis using an anti-yeast cytochrome c antibody.

[0032] [Bcl-x _L and interaction with the VDAC] (3 kinds of proteins that bind to Bcl-x _L) rat liver mitochondria (1 mg / ml) of recombinant Bcl-x
_L After incubation (20 [mu] g / ml) with 5 minutes, the mitochondrial lysate anti Bcl-x _L antibody (α-Bcl-x _L) or rabbit immunoglobulin G
The resulting immunocomplexes were subjected to immunoprecipitation using (IgG), separated by SDS-polyacrylamide gel electrophoresis, and stained by silver staining. This anti-Bcl
By co-immunoprecipitation with -x _L antibodies, three proteins that bind to Bcl-x _L in mitochondria,
33 kD, 18 kD, and 13 kD were detected (FIG. 1).
a).

[0033] 3 obtained by the above method (interaction with Bcl-x _L and VDAC mitochondrial)
Part of the amino acid sequence of the 3 kD protein is rat VD
AC and anti-VDAC antibody and anti-Bcl
Subsequently by Western blotting co immunoprecipitation with -x _L antibodies, B in isolated mitochondria
It examined the interaction with the cl-x _L and VDAC. The results are shown in FIG.

[0034] Hep expressing Bcl-x _L (interaction with Bcl-x _L and VDAC HepG2 cells)
B in G2 cells (Oncogene 12, 2045-2050, 1996)
It examined the interaction with the cl-x _L and VDAC. The results are shown in FIG. 1c. On the other hand, interactions can co-immunoprecipitation between Bcl-x _L and Tom40 (integral outer membrane protein) and Bcl-x _L and F ₁ -ATP-ase (inner membrane protein), because the observed together, Bcl-x _L and VDA
It turns out that C has a specific interaction.

(Bcl-x_LAnd VDAC or ANT
Direct interaction) PT pore is a polyprotease containing VDAC
Because it is a tein channel, mitochondria and
VDAC and Bcl-x from cells_LCo-immunoprecipitation in
But not necessarily Bcl-x_LDirect interaction between VDAC and VDAC
Since it cannot be said to show any effect, the following experiment was
I accepted. Purified rat liver VDAC (0.2 μg)
And rat heart ANT (0.2 μg) with GST-Bcl
-X_LFusion protein (0.2 μg) or GST (0.
2 μg), followed by
Lutathion (GSH)-Sepharose gel and incube
Was an option. GST-Bcl-x_LAnd Incubation
Separated gel with VDAC and ANT
And incubated. Then wash the gel 3 times and bind
Analyzed VDAC and ANT by Western blot
did. The results are shown in FIG. In FIG. 1d, “Total”
Represents the amount of each protein used for immunoprecipitation. Figure
Purified from rat liver mitochondria as shown in 1d
VDAC is GST-Bcl-x _LFusion protein
Bcl-x_LIs VDA
It was found to show a direct interaction with C. And
ANT purified from rat heart is GST-Bcl-
x_LShowed only a low percentage of binding to
Results from the literature (Science 281, 2027-2031, 1998)
I was doing it.

[VDAC-Bcl in liposome
VDAC activity inhibition by -x _L] (VDAC-sucrose uptake by liposomes) confirmed direct interaction between Bcl-x _L and VDAC from the above that, then, adjusting the direct VDAC activity Bcl-x _L is I checked whether to do. VDAC purified from rat liver mitochondrial homogenate was reconstituted in liposomes. In addition to this VDAC-liposome, heat-denatured VDAC-liposome and plain liposome as a control were used, and each of them was incubated with ¹⁴ C sucrose (1 μCi) at pH 5.2 for a predetermined time, and the molecular weight of the 30,000 restriction filter was reduced. After filtration by centrifugation using
It was dissolved in SDS and the radioactivity of ¹⁴ C sucrose in the liposome was measured. The results are shown in FIG. 2a. As shown in FIG. 2a, unlike heat-denatured VDAC-liposomes (□-□) and plain liposomes (●-●), VDAC-liposomes (○-○) incorporated sucrose labeled with a radioisotope. Showed that sucrose uptake of liposomes was mediated by VDAC.

(Bcl-x_LVDAC-Liposaw by
Of VDAC activity in the system)¹⁴C sucrose (1μC
20 μg of each VDAC-liposome using i)
/ Ml recombinant Bcl-x_L(○-○) or simulated tamper
And incubated at pH 5.2 with
Was. Similarly, recombinant Bcl-x_L(□-□) or mock-up
Incubate with protein (３- ■) at pH 7.3
did. These results are shown in FIG. 2b. smells pH 5.2
And Bcl-x_LWhen added, VDAC-mediated scrolling
Source uptake was inhibited. Bcl-x at acidic pH_LThe machine
The fact that it worked only on synthetic lipid membranes under acidic conditions
RBcl-x as ion channel _LLiterature that works
(Science 277, 370-372, 1997. Nature385, 353-357,
1997. Proc. Natl. Acad. Sci. USA 94, 5113-5118, 199
7. Proc. Natl. Acad. Sci. USA 94, 11357-11362, 199
It was consistent with the result of 7). Next, Bcl-x_LAnd VD
Consider the case where AC is simultaneously inserted into the liposome.
Was.

[0038] and (VDAC and inhibition of VDAC activity by Bcl-x _L at neutral pH in the liposomes containing the recombinant Bcl-x _L) Purified VDAC, recombinant B
The cl-x _L or VDAC / Bcl-x _L liposomes and VDAC / mock proteoliposomes introduced both mock proteins simultaneously into liposomes, incubated pH7.3 at 3 minutes with ¹⁴ C sucrose (1 [mu] Ci), ¹⁴ C sucrose The absorption was measured. The results are shown in FIG. 2c. As can be seen from FIG. 2c, even at pH 7.3, B
since it has a cl-x _L functions to suppress the uptake of ¹⁴ C sucrose, in an acidic environment it was suggested that the uptake of Bcl-x _L itself within liposomes is facilitated. It is also reported that VDAC is closed below pH 5.0, but the VDAC-liposomes in this experiment did not show any significant difference in behavior between pH 5.2 and 7.3.

[0039] (VD in various Bcl-x _L dose
Inhibition of AC activity and Bcl-x _L VD by mutant
Inhibition of AC activity) Next, 0, 10, 20 and 50 μm
g / ml of recombinant Bcl-x _L and 20 μg / m each.
l of recombinant Bcl-x _L mutant mt-1 (F131V,
D133A) and mt-7 (135-137 is using ¹⁴ C-sucrose (1 [mu] Ci) with VNW) from AIL, after incubation with 3 minutes VDAC- liposome pH 5.2, was measured ¹⁴ C-sucrose in the liposomes. The results are shown in FIG. 2d. As shown in FIG.
It can be seen that the inhibition of VDAC activity increases with increasing amount of l-x _L increases. In addition, two types of recombinant Bc
Among l-x _L mutant, the mt-1 has a partially anti-apoptotic activity, it is literature mt-7 does not have any anti-apoptotic activity (Nature 379, 554-556, 199
As shown in 6), with respect to VDAC-mediated sucrose absorption, these mutants each had a partial inhibitory effect on mt-1 and no inhibitory effect on mt-7. The two activities were found to be correlated.

[0040] The (inhibition of VDAC activity by Bcl-x _L in liposomes incorporating the recombinant VDAC) purified recombinant VDAC, incorporated into liposomes, then ¹⁴
Incubated C sucrose (1 [mu] Ci) and of recombinant _{Bcl-x L (20μg / ml} ) with pH5.2 in 3 minutes was measured ¹⁴ C-sucrose uptake. Figure 2 shows the results.
e. If the VDAC- liposomes using recombinant VDAC made, since the inhibition of VDAC activity was also observed by Bcl-x _L, VDAC is Bcl-x
_L was confirmed to be a direct target.

[Inhibition of VDAC Activity by rBcl-x _L and Promotion of VDAC Activity by rBax]
It was examined whether or not affected VDAC activity. (Promotion of Incorporation of ¹⁴ C Sucrose into VDAC-Liposome by Bax) Using ¹⁴ C sucrose (1 μCi), VDAC-liposome was reconstituted with 20 μg / ml of recombinant Bax (（-○) or mock protein (●-●). )
For a predetermined time at pH 5.2.
Similarly, the VDAC-liposomes were changed to plain liposomes and incubated with recombinant Bax (□-□) or mock protein (■-■), resulting in a molecular weight of 30,00.
After filtration by centrifugation using a 0 restriction filter,
After the liposome was collected and dissolved in 2% SDS, the radioactivity of ¹⁴ C sucrose in the liposome was measured. The results are shown in FIG. 3a. FIG. 3a shows that rBax increased VDAC-mediated sucrose uptake, but no uptake of Bax itself into plain liposomes. Thus, the increase in rBax-induced sucrose uptake in VDAC-liposomes is not mediated by Bax ion channels,
9 shows that it was mediated through some action of Bax in DAC.

(Promotion of VDAC Activity by Bax in Liposome Incorporating Recombinant VDAC) Purified recombinant VDAC was incorporated into liposome,
ax (20 μg / ml) or ¹⁴ C with mock protein
Sucrose (1 μCi) was used to incubate at pH 5.2 and ¹⁴ C sucrose absorption was measured for 3 minutes. The results are shown in FIG. As can be seen from FIG. 3b, similar results were observed when VDAC-liposomes were prepared using recombinant VDAC.

(VD by rBax and rBcl-x _L
Antagonism for sucrose import into AC liposomes) rBax and rBc in proportions shown in FIG. 3c
with l-x _L, ¹⁴ C sucrose (1 [mu] Ci), VDA
C-liposomes were used to incubate at pH 5.2 and ¹⁴ C sucrose absorption was measured for 3 minutes. Fig. 3 shows the results.
c. As shown in FIG. 3c, rBax and Bcl
The -x _L, exhibited antagonism in VDAC activity.

(Direct interaction between Bax and VDAC)
When both rBax and VDAC were incorporated into liposomes, Bax exhibited an apoptosis-promoting action at pH 7.3, and a mutant lacking the C-terminal 21 amino acid residues of Bak having an apoptosis-promoting action instead of rBax was used. Similar results were obtained when used. Anti-VD
When immunocomplexes were analyzed by Western blotting using the AC antibody, VDAC prepared from rat liver and E. coli did not contain detectable levels of ANT, and thus were two more ANT ligands. VDAC- by atractylic acid and boncrekinic acid
Sucrose uptake by liposomes was not affected. From this it can be concluded that there was no ANT contamination in the VDAC preparation.

[Induction of cytochrome c through VDAC by Bax and Bak] The present inventors have already reported the PT pore (Proc. Natl. Acad. Sci. USA 95, 4997-5002, 199).
8. Proc. Natl. Acad. Sci. USA 95, 14681-14686, 199
8) that Bax and Bak induce the release of cytochrome c from isolated mitochondria (Pr
oc. Natl. acad. Sci. USA 95, 4997-5002, 1998. J. C
ell Biol. 143, 217-224, 1998. Proc. Natl. acad. Sc
i. USA 95, 14681-14686, 1998). As described above, since Bax and Bak were found to directly increase VDAC activity, it was examined whether cytochrome c is released through VDAC in the presence of Bax and Bak.

(FITC Label of Cytochrome c) In order to measure the movement of cytochrome c using a microscope, FITC-labeled cytochrome c was prepared so as not to make a significant change in its molecular mass, and was subjected to SDS-polyacrylamide gel electrophoresis. Analyzed by electrophoresis. FITC was detected by fluorescence, and cytochrome c was detected by Western blotting using an anti-cytochrome c antibody. In FIG. 4a, lane 1 shows FITC-cytochrome c (25 pg), and lane 2 shows cytochrome c (25 pg).

(Incorporation of FITC-cytochrome c into VDAC-liposome by Bax induction) As described above, when 50 μM FITC-cytochrome c and 50 mM sucrose were used, and when rBax (52 μg / ml) was used, rBax and rBcl-x _L (both 52 μg
/ Ml) or when neither is used
VDAC-liposomes (Fig. 4b, 4
c) and plain liposomes (FIG. 4c) were incubated and observed under a confocal fluorescence microscope. A typical photograph (4,800 times) of the VDAC-liposome is shown in FIG. 4b. Liposomes were classified based on cytochrome c accumulation in the liposome and the frequency (%) and a typical photograph are shown in FIG.
Shown in Each photograph is 2 μm × 2 μm.

As shown in FIGS. 4b and 4c, the VDAC-liposome was incubated with FITC-labeled cytochrome c in the presence of sucrose to detect labeled cytochrome c under a confocal fluorescence microscope. As a result, in the absence of rBax, cytochrome c was accumulated on the surface of the plain liposome as in the case of the VDAC-liposome (FIGS. 4b and 4c). In contrast, rBax (FIG. 4b,
4c) In the presence of cytochrome c accumulated inside VDAC-liposome, Bax added VD to cytochrome c.
It was found to allow AC passage. rBax and rBcl
The presence of -x _L, moved by Bax-mediated cytochrome c was inhibited (Fig. 4b, 4c). When Bak was used instead of rBax, similar results were obtained.

(VDAC-Bak-Induced Cytochrome c Release from Liposomes) As described above, VDAC-containing 80 mM sucrose and 100 μM cytochrome c
Liposomes (left panel) and plain liposomes (right panel) were incubated for 5 minutes at pH 5.2 in sucrose-free buffer in the presence or absence of rBak (20 μg / ml). The results are shown in FIG. 4d. FIG. 4d shows that the total amount of cytochrome c initially taken in is 100%.
Shown as%. Cytochrome c release was measured with a spectrophotometer.

As can be seen from FIG. 4d, cytochrome c and sucrose were incorporated into the VDAC-liposome, and then VDAC-liposome was added using a sucrose-free buffer.
When the liposomes were incubated, cytochrome c was released in a large amount in the presence of rBak, but rBa was released.
No such cytochrome c release was observed when plain liposomes were used in the presence of x. Also, the small amount of cytochrome c release from both VDAC-liposomes and plain liposomes after incubation with the sucrose-free buffer is thought to be due to the release of non-specifically bound cytochrome c on the liposome surface. Can be

In the absence of sucrose, it was also observed that Bak allowed cytochrome c to permeate the VDAC. During these experiments, the number of liposomes remained unchanged, as measured by flow cytometry, with GST
-VDAC containing green fluorescent protein (GFP)-
When rBak was added to liposomes, GST-
The GFP fusion protein (50 kD) was not released, indicating that liposome rupture did not occur. Also, it was found that when rBax was used instead of rBak, cytochrome c was similarly transmitted through VDAC.

[VDAC for Bak-Induced ΔΨ Loss and Cytochrome c Release in Yeast Mitochondria]
Requirement] Since Bcl-2 family proteins were found to target VDAC directly to mediate channel activity, VDAC then released mitochondrial ΔΨ loss and cytochrome c release associated with apoptosis. It was examined whether the target was a target of Bcl-2 family proteins involved in the regulation of E. coli.

(RDAC of VDAC-deficient mitochondria)
Inability to induced ΔΨ loss) VDAC-deficient cells from mammals are not available and Bcl-2 family proteins have been shown to function in yeast cells (FEBS lett. 380, 169-175, 1996. Molecular C
ell 1, 327-336, 1998), and examined using VDAC1 deleted ΔVDAC (Science 247, 1233-1236, 1990). rBak (50 μg / ml) (FIG. 5a), mock protein (50 μg / ml) (FIG. 5b), and rB
ak (50 μg / ml) and rBcl-x _L (20 μg / ml)
ml) (FIG. 5c) using wild-type yeast (wild),
Mitochondria (0.5 mg / ml) each isolated from ΔVDAC yeast (hVDAC) expressing ΔVDAC and human VDAC were incubated with Rh123 and Δ 、 was measured. The results are shown in FIGS.

(VDAC1-deficient mitochondria without Bak-induced cytochrome c release) rBcl-x
RBak in the presence or absence of _L (20 μg / ml)
(50 μg / ml) or mock protein (50 μg / m
l) for 20 minutes using wild-type yeast (wild), Δ
ΔVDAC yeast expressing VDAC and hVDAC (h
Mitochondria (0.5 mg isolated from VDAC)
/ Ml) was incubated. Samples were centrifuged and a portion of the supernatant (20 μl) was analyzed by Western blot using an anti-yeast cytochrome c antibody. The results are shown in FIG. 5d. “Total” in FIG. 5d represents an equivalent amount of mitochondria.

As can be seen from FIGS. 5a and 5d, mitochondria isolated from wild-type yeast, as well as mitochondria from mammals, showed ΔΨ loss and cytochrome c release after addition of rBak, while ΔVDAC yeast No induction of ΔΨ loss and cytochrome c release by rBax was observed in mitochondria isolated from E. coli. However, the human vdac1 gene (hVDA
In the mitochondria from the ΔVDAC yeast transfected with C), the fact that the mitochondria from the ΔVDAC yeast did not respond to rBak exists because rBak induced ΔΨ loss and cytochrome c release. It turned out that it was not.

[0056] As can be seen from FIGS. 5c and 5d, in yeast mitochondria, Bak-mediated ΔΨ loss and cytochrome c release was inhibited by Bcl-x _L. The mitochondria used in this study all had the same level of ATP, thus excluding potential effects due to qualitative differences in the mitochondria used. Bcl
It is clear from this result that the -2 family proteins target VDAC and regulate mitochondrial ΔΨ loss and cytochrome c release associated with apoptosis.

[0057]

According to the method for screening an apoptosis-suppressing substance or an apoptosis-promoting substance of the present invention, an apoptosis-suppressing substance or an apoptosis-promoting substance which can be expected to be used as a drug or a diagnostic agent in various diseases caused by an apoptosis-suppressing action or an apoptosis-promoting action. Can be provided. Further, the apoptosis-suppressing substance or the apoptosis-promoting substance of the present invention can be expected to be used as a medicine or a diagnostic agent in various diseases caused by an apoptosis-suppressing action or an apoptosis-promoting action.

[0058]

[Sequence List] SEQUENCE LISTING <110> Japan Science and Technology Corporation <120> Processes for screening apoptotic inhibitor or activator <130> A081P03 <160> 6 <170> PatentIn Ver. 2.0 <210> 1 <211> 771 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (32) .. (751) <223> Human Bcl-2 protein <400> 1 gttggccccc gttacttttc ctctgggaaa t atg gcg cac gct ggg aga aca 52 Met Ala His Ala Gly Arg Thr 1 5 ggg tac gat aac cgg gag ata gtg atg aag tac atc cat tat aag ctg 100 Gly Tyr Asp Asn Arg Glu Ile Val Met Lys Tyr Ile His Tyr Lys Leu 10 15 20 tcg cag agg ggc tac gag tgg gat gcg gga gat gtg ggc gcc gcg ccc 148 Ser Gln Arg Gly Tyr Glu Trp Asp Ala Gly Asp Val Gly Ala Ala Pro 25 30 35 ccg ggg gcc gcc ccc gcg ccg ggc atc ttc tcc tcg cag ccc ggg cac Ala Pro 196 G Pro Ala Pro Gly Ile Phe Ser Ser Gln Pro Gly His 40 45 50 55 acg ccc cat aca gcc gca tcc cgg gac ccg gtc gcc agg acc tcg ccg 244 Thr Pro His Thr Ala Ala Ser Arg Asp Pro Val Ala Arg Thr Ser Pro 60 65 70 ctg cag acc ccg gct gcc ccc ggc gcc gcc gcg ggg cct gcg ctc agc 292 Leu Gln Thr Pro Ala Ala Pro Gly Ala Ala Ala Gly Pro Ala Leu Ser 75 80 85 ccg gtg cca cct gtg gtc cac ctg acc ctc cgc cag gcc ggc gac gac 340 Pro Val Pro Val Val His Leu Thr Leu Arg Gln Ala Gly Asp Asp 90 95 100 ttc tcc cgc cgc tac cgc cgc gac ttc gcc gag atg tcc agg cag ctg 388 Phe Ser Arg Arg Tyr Arg Arg Asp Phe Ala Glu Met Ser Arg Gln Leu 105 110 115 cac ctg acg ccc ttc acc gcg cgg gga cgc ttt gcc acg gtg gtg gag 436 His Leu Thr Pro Phe Thr Ala Arg Gly Arg Phe Ala Thr Val Val Glu 120 125 130 135 gag ctc ttc agg gac ggg gtg aac tgg ggg agg att gt gcc ttc ttt 484 Glu Leu Phe Arg Asp Gly Val Asn Trp Gly Arg Ile Val Ala Phe Phe 140 145 150 gag ttc ggt ggg gtc atg tgt gtg gag agc gtc aac cgg gag atg tcg 532 Glu Phe Gly Glylu Met Cys Val Val Asn Arg Glu Met Ser 155 160 165 ccc ctg gtg gac aac atc gcc ctg tgg atg act gag tac ctg aac cgg 580 Pro Leu Val Asp Asn Ile Ala Leu Trp Met Thr Glu Tyr Leu Asn Arg 170 175 180 cac ctg cac acc tgg atc cag gat aac gga ggc tgg gat gcc ttt gtg 628 His Leu His Thr Trp Ile Gln Asp Asn Gly Gly Trp Asp Ala Phe Val 185 190 195 gaa ctg tac ggc ccc agc atg cgg cct ctg ttt gat ttc Tcc Ggg ctg yr Gly Pro Ser Met Arg Pro Leu Phe Asp Phe Ser Trp Leu 200 205 210 215 tct ctg aag act ctg ctc agt ttg gcc ctg gtg gga gct tgc atc acc 724 Ser Leu Lys Thr Leu Leu Ser Leu Ala Leu Val Gly Ala Cys Ile Thr 220 225 230 ctg ggt gcc tat ctg ggc cac aag tga agtcaacatg cctgccccaa 771 Leu Gly Ala Tyr Leu Gly His Lys 235 240 <210> 2 <211> 239 <212> PRT <213> Homo sapiens <400> 2 Met Ala His Ala Gly Arg Thr Gly Tyr Asp Asn Arg Glu Ile Val Met 1 5 10 15 Lys Tyr Ile His Tyr Lys Leu Ser Gln Arg Gly Tyr Glu Trp Asp Ala 20 25 30 Gly Asp Val Gly Ala Ala Pro Pro Gly Ala Ala Pro Ala Pro Gly Ile 35 40 45 Phe Ser Ser Gln Pro Gly His Thr Pro His Thr Ala Ala Ser Arg Asp 50 55 60 Pro Val Ala Arg Thr Ser Pro Leu Gln Thr Pro Ala Ala Pro Gly Ala 65 70 75 80 Ala Ala Gly Pro Ala Leu Ser Pro Val Pro Pro Val Val His Leu T hr 85 90 95 Leu Arg Gln Ala Gly Asp Asp Phe Ser Arg Arg Tyr Arg Arg Asp Phe 100 105 110 Ala Glu Met Ser Arg Gln Leu His Leu Thr Pro Phe Thr Ala Arg Gly 115 120 125 Arg Phe Ala Thr Val Val Glu Glu Leu Phe Arg Asp Gly Val Asn Trp 130 135 140 Gly Arg Ile Val Ala Phe Phe Glu Phe Gly Gly Val Met Cys Val Glu 145 150 155 160 Ser Val Asn Arg Glu Met Ser Pro Leu Val Asp Asn Ile Ala Leu Trp 165 170 175 Met Thr Glu Tyr Leu Asn Arg His Leu His Thr Trp Ile Gln Asp Asn 180 185 190 Gly Gly Trp Asp Ala Phe Val Glu Leu Tyr Gly Pro Ser Met Arg Pro 195 200 205 Leu Phe Asp Phe Ser Trp Leu Ser Leu Lys Thr Leu Leu Ser Leu Ala 210 215 220 Leu Val Gly Ala Cys Ile Thr Leu Gly Ala Tyr Leu Gly His Lys 225 230 235 <210> 3 <211> 926 <212> DNA <213> Homo sapiens <220> <221> CDS < 222> (135) .. (836) <223> Human Bcl-xL protein <400> 3 gaatctcttt ctctcccttc agaatcttat cttggctttg gatcttagaa gagaatcact 60 aaccagagac gagactcagt gagtgagcag gtgttttgga caatggactg gttgagcccag attgagcag tttgagcccag ag gtg gtt gac ttt 170 Met Ser Gln Ser Asn Arg Glu Leu Val Val Asp Phe 1 5 10 ctc tcc tac aag ctt tcc cag aaa gga tac agc tgg agt cag ttt agt 218 Leu Ser Tyr Lys Leu Ser Gln Lys Gly Tyr Ser Trp Ser Gln Phe Ser 15 20 25 gat gtg gaa gag aac agg act gag gcc cca gaa ggg act gaa tcg gag 266 Asp Val Glu Glu Asn Arg Thr Glu Ala Pro Glu Gly Thr Glu Ser Glu 30 35 40 atg gag acc ccc agt gcc atc aat ggc aac cca tcc tgg cac ctg gca 314 Met Glu Thr Pro Ser Ala Ile Asn Gly Asn Pro Ser Trp His Leu Ala 45 50 55 60 gac agc ccc gcg gtg aat gga gcc act gcg cac agc agc agt ttg gat 362 Asp Ser Pro Ala Val Asn Gly Ala Thr Ala His Ser Ser Ser Leu Asp 65 70 75 gcc cgg gag gtg atc ccc atg gca gca gta aag caa gcg ctg agg gag 410 Ala Arg Glu Val Ile Pro Met Ala Ala Val Lys Gln Ala Leu Arg Glu 80 85 90 gca ggc gac gag ttt gaa ctg cgg tac cgg cgg gca ttc agt gac ctg 458 Ala Gly Asp Glu Phe Glu Leu Arg Tyr Arg Arg Ala Phe Ser Asp Leu 95 100 105 aca tcc cag ctc cac atc acc cca ggg aca gca tat agc ttt gaa 506 Thr Ser Gln Leu His Ile Thr Pro Gly Thr Ala Tyr Gln Ser Phe Glu 110 115 120 cag gta gtg aat gaa ctc ttc cgg gat ggg gta aac tgg ggt cgc att 554 Gln Val Val Asn Glu Leu Phe Arg Asp Gly Val Asn Trp Gly Arg Ile 125 130 135 140 gtg gcc ttt ttc tcc ttc ggc ggg gca ctg tgc gtg gaa agc gta gac 602 Val Ala Phe Phe Ser Phe Gly Gly Ala Leu Cys Val Glu Ser Val Asp 145 150 155 aag gag atg cag gta ttg gtg atc gca gct tgg atg gcc act 650 Lys Glu Met Gln Val Leu Val Ser Arg Ile Ala Ala Trp Met Ala Thr 160 165 170 tac ctg aat gac cac cta gag cct tgg atc cag gag aac ggc ggc tgg 698 Tyr Leu Asn Asp His Leu Glu Pro Trp Ile Gln Glu Asn Gly Gly Trp 175 180 185 gat act ttt gtg gaa ctc tat ggg aac aat gca gca gcc gag agc cga 746 Asp Thr Phe Val Glu Leu Tyr Gly Asn Asn Ala Ala Ala Glu Ser Arg 190 195 200 aag ggc cag gaa cgc ttc aac cgc tgg ttc ctg acg ggc atg act gtg 794 Lys Gly Gln Glu Arg Phe Asn Arg Trp Phe Leu Thr Gly Met Thr Val 205 210 215 220 gcc ggc gtg gtt ctg ctg ggc tcagt836 Ala Gly Val Val Leu Leu Gly Ser Leu Phe Ser Arg Lys 225 230 ccagacactg accatccact ctaccctccc acccccttct ctgctccacc acatcctccg 896 tccagccgcc attgccacca ggagaacccg 926 <210> 4 <211> 233 <212> PRT 213 <212> PRT Gln Ser Asn Arg Glu Leu Val Val Asp Phe Leu Ser Tyr Lys 1 5 10 15 Leu Ser Gln Lys Gly Tyr Ser Trp Ser Gln Phe Ser Asp Val Glu Glu 2025 30 Asn Arg Thr Glu Ala Pro Glu Gly Thr Glu Ser Glu Met Glu Thr Pro 35 40 45 Ser Ala Ile Asn Gly Asn Pro Ser Trp His Leu Ala Asp Ser Pro Ala 50 55 60 Val Asn Gly Ala Thr Ala His Ser Ser Ser Leu Asp Ala Arg Glu Val 65 70 75 80 Ile Pro Met Ala Ala Val Lys Gln Ala Leu Arg Glu Ala Gly Asp Glu 85 90 95 Phe Glu Leu Arg Tyr Arg Arg Ala Phe Ser Asp Leu Thr Ser Gln Leu 100 105 110 His Ile Thr Pro Gly Thr Ala Tyr Gln Ser Phe Glu Gln Val Val Asn 115 120 125 Glu Leu Phe Arg Asp Gly Val Asn Trp Gly Arg Ile Val Ala Phe Phe 130 135 140 Ser Phe Gly Gly Ala Leu Cys Val Glu Ser Val Asp Lys Glu Met Gln 145 150 155 160 Val Leu Val Ser Arg Ile Ala Ala Trp Met Ala Thr Tyr Leu Asn Asp 165 170 175 His Leu Glu Pro Trp Ile Gln Glu Asn Gly Gly Trp Asp Thr Phe Val 180 185 190 Glu Leu Tyr Gly Asn Asn Ala Ala Ala Glu Ser Arg Lys Gly Gln Glu 195 200 205 Arg Phe Asn Arg Trp Phe Leu Thr Gly Met Thr Val Ala Gly Val Val 210 215 220 Leu Leu Gly Ser Leu Phe Ser Arg Lys 225 230 <210> 5 <211> 579 <212> DNA <213> Homo sapiens <220 > <221> CDS <222> (1) .. (579) <223> Human Bax protein <400> 5 atg gac ggg tcc ggg gag cag ccc aga ggc ggg ggg ccc acc agc tct 48 Met Asp Gly Ser Gly Glu Gln Pro Arg Gly Gly Gly Gly Pro Thr Ser Ser 1 5 10 15 gag cag atc atg aag aca ggg gcc ctt ttg ctt cag ggt ttc atc cag 96 Glu Gln Ile Met Lys Thr Gly Ala Leu Leu Leu Gln Gly Phe Ile Gln 20 25 30 gat cga gca ggg cga atg ggg ggg gag gca ccc gag ctg gcc ctg gac 144 Asp Arg Ala Gly Arg Met Gly Gly Glu Ala Pro Glu Leu Ala Leu Asp 35 40 45 ccg gtg cct cag gat gcg tcc acc aag aag ctg agc gag aag 192 Pro Val Pro Gln Asp Ala Ser Thr Lys Lys Leu Ser Glu Cys Leu Lys 50 55 60 cgc atc ggg gac gaa ctg gac agt aac atg gag ctg cag agg atg att 240 Arg Ile Gly Asp Glu Leu Asp Ser Asn Met Glu Leu Gln Arg Met Ile 65 70 75 80 gcc gcc gtg gac aca gac tcc ccc cga gag gtc ttt cga gtg gca 288 Ala Ala Val Asp Thr Asp Ser Pro Arg Glu Val Phe Phe Arg Val Ala 85 90 95 gct gac atg ttt tct gac ggc aac ttc aac tgg ggc cgg gtt gtc gcc 336 Ala Asp Met Phe Ser Asp Gly Asn Phe Asn Trp Gly Arg Val Val Ala 100 105 110 ctt ttc tac ttt gcc agc aaa ctg gtg ctc aag gcc ctg tgc acc aag 384 Leu Phe Tyr Phe Ala Ser Lys Leu Val Leu Lys Ala Leu Cys Thr Lys 115 120 125 gtg ccg gaa ctg aga acc atc atg ggc tgg aca ttg gac ttc ctc 432 Val Pro Glu Leu Ile Arg Thr Ile Met Gly Trp Thr Leu Asp Phe Leu 130 135 140 cgg gag cgg ctg ttg ggc tgg atc caa gac cag ggt ggt tgg gac ggc 480 Arg Arg Leu Leu Gly Trp Ile Gln Asp Gln Gly Gly Trp Asp Gly 145 150 155 160 ctc ctc tcc tac ttt ggg acg ccc acg tgg cag acc gtg acc atc ttt 528 Leu Leu Ser Tyr Phe Gly Thr Pro Thr Trp Gln Thr Val Thr Thr Ile Phe 16 5 170 175 gtg gcg gga gtg ctc acc gcc tcg ctc acc atc tgg aag aag atg ggc 576 Val Ala Gly Val Leu Thr Ala Ser Leu Thr Ile Trp Lys Lys Met Gly 180 185 190 tga 579 <210> 6 <211> 192 < 212> PRT <213> Homo sapiens <400> 6 Met Asp Gly Ser Gly Glu Gln Pro Arg Gly Gly Gly Pro Thr Ser Ser 1 5 10 15 Glu Gln Ile Met Lys Thr Gly Ala Leu Leu Leu Gln Gly Phe Ile Gln 20 25 30 Asp Arg Ala Gly Arg Met Gly Gly Glu Ala Pro Glu Leu Ala Leu Asp 35 40 45 Pro Val Pro Gln Asp Ala Ser Thr Lys Lys Leu Ser Glu Cys Leu Lys 50 55 60 Arg Ile Gly Asp Glu Leu Asp Ser Asn Met Glu Leu Gln Arg Met Ile 65 70 75 80 Ala Ala Val Asp Thr Asp Ser Pro Arg Glu Val Phe Phe Arg Val Ala 85 90 95 Ala Asp Met Phe Ser Asp Gly Asn Phe Asn Trp Gly Arg Val Val Ala 100 105 110 Leu Phe Tyr Phe Ala Ser Lys Leu Val Leu Lys Ala Leu Cys Thr Lys 115 120 125 Val Pro Glu Leu Ile Arg Thr Ile Met Gly Trp Thr Leu Asp Phe Leu 130 135 140 Arg Glu Arg Leu Leu Gly Trp Ile Gln Asp Gln Gly Gly Trp Asp Gly 145 150 155 160 Leu Leu Ser Tyr Phe Gly Thr Pro T hr Trp Gln Thr Val Thr Ile Phe 165 170 175 Val Ala Gly Val Leu Thr Ala Ser Leu Thr Ile Trp Lys Lys Met Gly 180 185 190

[Brief description of the drawings]

FIG. 1 shows the interaction between Bcl-x _L and VDAC.

FIG. 2: Bcl-x _L in VDAC-liposome system
FIG. 4 is a diagram showing inhibition of VDAC activity by VDAC.

FIG. 3. Promotion of VDAC activity by rBax and rBcl
FIG. 4 shows the inhibition of VDAC activity by −x _L.

FIG. 4. VDA of cytochrome c by Bax and Bak.
It is a figure showing guidance of C passage.

FIG. 5. Bak induction ΔΨ in yeast mitochondria.
FIG. 4 is a diagram illustrating VDAC requirements for loss and cytochrome c release.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C07K 14/705 C12P 21/02 C 4H045 C12N 5/10 G01N 33 / 15Z C12P 21/02 33 / 50Z G01N 33/15 A61K 37/02 33/50 C12N 5/00 BF term (reference) 2G045 AA25 AA34 AA35 AA40 BB20 DA12 DA13 DA14 DA36 DA77 FB02 FB07 4B024 AA11 BA80 CA04 DA02 DA03 EA04 GA11 GA30 HA20 4B064 AG01 CA02 CA02 DA03 DA13 4B065 AA26X AA93Y AB01 AC14 BA02 BA16 CA24 CA44 CA46 4C084 AA07 AA17 BA01 BA18 BA19 ZB212 4H045 AA10 BA10 BA17 BA18 CA40 EA20 EA50 FA72 FA74

Claims (26)

[Claims] 1. Incubating a VDAC-liposome, an indicator substance capable of passing through the VDAC, and a test substance, the VDA before and after the incubation.
C-A screening method for an apoptosis-suppressing substance or an apoptosis-promoting substance, which comprises determining the presence or absence of an apoptosis-suppressing action or an apoptosis-promoting action of a test substance by detecting a change in the concentration of an indicator substance inside and outside the liposome. 2. Incubation is carried out when the indicator substance is VD.
The method for screening an apoptosis-suppressing substance or apoptosis-promoting substance according to claim 1, wherein the method is performed in a state where the substance is present in an AC-liposome. 3. The method according to claim 1, wherein the test substance is VD.
The method for screening an apoptosis-suppressing substance or apoptosis-promoting substance according to claim 1 or 2, wherein the method is performed in a state where the substance is present in an AC-liposome. 4. The method for screening an apoptosis-suppressing substance or an apoptosis-promoting substance according to claim 1 or 2, wherein the incubation is performed in a state where the apoptosis-suppressing substance or the apoptosis-promoting substance is present together with the test substance. 5. The method of claim 5, wherein the VDAC-liposome is a recombinant human V-liposome.
The method for screening for an apoptosis-suppressing substance or an apoptosis-promoting substance according to any one of claims 1 to 4, which is a VDAC-liposome prepared using DAC. 6. The method for screening an apoptosis-suppressing substance or apoptosis-promoting substance according to any one of claims 1 to 5, wherein the VDAC-liposome is a VDAC-liposome obtained by reconstituting VDAC into small unilamellar vesicles. . 7. The method for screening an apoptosis-suppressing substance or apoptosis-promoting substance according to claim 1, wherein the indicator substance is a labeled compound. 8. The method according to claim 7, wherein the labeled compound is fluorescently labeled cytochrome c and / or isotope-labeled sucrose. 9. The method for screening an apoptosis-suppressing substance or apoptosis-promoting substance according to any one of claims 1 to 8, wherein the test substance is a protein or a polypeptide. 10. The method according to claim 10, wherein the protein or polypeptide is Bc
1 in the amino acid sequence of the 1-2 family protein
10. The method for screening an apoptosis-suppressing substance or apoptosis-promoting substance according to claim 9, which is a protein or polypeptide having an amino acid sequence in which several amino acids have been deleted, substituted or added. 11. An amino acid in which a protein or polypeptide has one or several amino acids deleted, substituted or added in a polypeptide having an amino acid sequence of the 7th to 30th amino acids in the amino acid sequence shown in SEQ ID NO: 2. 10. The method for screening an apoptosis-suppressing substance or apoptosis-promoting substance according to claim 9, which is a protein or polypeptide containing a sequence. 12. An amino acid in which a protein or polypeptide has one or several amino acids deleted, substituted or added in a polypeptide having the 4th to 23rd amino acid sequences of the amino acid sequence shown in SEQ ID NO: 4. 10. The method for screening an apoptosis-suppressing substance or apoptosis-promoting substance according to claim 9, which is a protein or polypeptide containing a sequence. 13. A protein or polypeptide comprising the amino acid sequence shown in SEQ ID NO: 6 from the 55th position to the 74th position.
10. The apoptosis-suppressing substance or apoptosis-promoting substance according to claim 9, wherein the polypeptide has an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the polypeptide having the amino acid sequence. A method for screening substances. 14. The method according to claim 14, wherein the protein or polypeptide is Bc
A protein or polypeptide obtained by using, as a probe, a nucleotide sequence of DNA encoding a 1-2 family protein or its complementary sequence, and a DNA containing a part or all of these sequences. 10. The method for screening an apoptosis-suppressing substance or an apoptosis-promoting substance according to 9. 15. A protein or polypeptide comprising a base sequence represented by SEQ ID NO: 1 from the 32nd position to the 751th position.
The apoptosis-inhibiting substance according to claim 9, comprising a protein or polypeptide obtained by using as a probe a DNA containing the nucleotide sequence up to the second or its complementary sequence and a part or all of these sequences. A method for screening an apoptosis promoting substance. 16. The protein or polypeptide is selected from the base sequence shown in SEQ ID NO: 3 from position 135 to position 83.
10. The apoptosis-suppressing substance according to claim 9, comprising a protein or a polypeptide obtained by using as a probe a DNA containing up to the sixth base sequence or its complementary sequence and a part or all of these sequences. Or a method for screening an apoptosis promoting substance. 17. The protein or polypeptide is selected from the base sequence shown in SEQ ID NO: 5 from the 163rd position to the 22nd position.
10. The apoptosis-suppressing substance according to claim 9, comprising a protein or a polypeptide obtained by using as a probe a DNA containing up to the second nucleotide sequence or its complementary sequence and a part or all of these sequences. Or a method for screening an apoptosis promoting substance. 18. A polypeptide having the 7th to 30th amino acid sequence of the amino acid sequence shown in SEQ ID NO: 2. 19. An apoptosis-suppressing action, which comprises at least one amino acid sequence of the 7th to 30th amino acids in the amino acid sequence represented by SEQ ID NO: 2 in which one or several amino acids are deleted, substituted or added. Polypeptide. 20. A polypeptide having the 4th to 23rd amino acid sequences of the amino acid sequence shown in SEQ ID NO: 4. 21. An apoptosis-suppressing activity, which comprises at least one amino acid sequence of the amino acid sequence of SEQ ID NO: 4 in which one or several amino acids are deleted, substituted or added in the fourth to 23rd amino acid sequences. Polypeptide. 22. A polypeptide having the amino acid sequence of positions 55 to 74 in the amino acid sequence shown in SEQ ID NO: 6. 23. In the amino acid sequence represented by SEQ ID NO: 6, 1 in the 55th to 74th amino acid sequences
Alternatively, a polypeptide having an apoptosis-promoting activity, comprising at least an amino acid sequence in which several amino acids have been deleted, substituted or added. 24. The polypeptide according to claim 18, wherein the polypeptide is chemically modified for introduction into a cell.
The polypeptide according to any one of the above. An apoptosis-suppressing substance obtained by the screening method according to any one of claims 1 to 17. An apoptosis-promoting substance obtained by the screening method according to any one of claims 1 to 17.